Three Yersinia pestis Adhesins Facilitate Yop Delivery to Eukaryotic Cells and Contribute to Plague Virulence

To establish a successful infection, Yersinia pestis requires the delivery of cytotoxic Yops to host cells. Yops inhibit phagocytosis, block cytokine responses, and induce apoptosis of macrophages. The Y. pestis adhesin Ail facilitates Yop translocation and is required for full virulence in mice. To determine the contributions of other adhesins to Yop delivery, we deleted five known adhesins of Y. pestis. In addition to Ail, plasminogen activator (Pla) and pH 6 antigen (Psa) could mediate Yop translocation to host cells. The contribution of each adhesin to binding and Yop delivery was dependent upon the growth conditions. When cells were pregrown at 28°C and pH 7, the order of importance for adhesins in cell binding and cytotoxicity was Ail > Pla > Psa. Y. pestis grown at 37°C and pH 7 had equal contributions from Ail and Pla but an undetectable role for Psa. At 37°C and pH 6, both Ail and Psa contributed to binding and Yop delivery, while Pla contributed minimally. Pla-mediated Yop translocation was independent of protease activity. Of the three single mutants, the Δail mutant was the most defective in mouse virulence. The expression level of ail was also the highest of the three adhesins in infected mouse tissues. Compared to an ail mutant, additional deletion of psaA (encoding Psa) led to a 130,000-fold increase in the 50% lethal dose for mice relative to that of the KIM5 parental strain. Our results indicate that in addition to Ail, Pla and Psa can serve as environmentally specific adhesins to facilitate Yop secretion, a critical virulence function of Y. pestis.The causative agent of plague is the Gram-negative bacterium Yersinia pestis (54, 77). Plague is one of the most deadly infectious diseases and has decimated civilizations repeatedly throughout history (10, 54). Plague still remains a public health concern, and due to the increasing number of cases worldwide, plague is classified as a reemerging infectious disease (68). Identification of therapies or effective vaccines would provide protection against plague as a potential bioterrorism threat.There are three clinical forms of plague in humans: bubonic, pneumonic, and septicemic (54). Bubonic plague is the most common form and usually occurs following a fleabite. In bubonic plague, the organism initially spreads to the regional lymph nodes, where it replicates primarily extracellularly, and then eventually enters the bloodstream. If untreated, bubonic plague is fatal in 40 to 60% of cases (54). Pneumonic plague is the least common form, but it progresses rapidly and is the most fatal form of the disease. Pneumonic plague may occur as a complication of bubonic or septicemic plague (secondary pneumonic plague) or by inhalation of infectious droplets spread by the cough or sneeze of a person with pneumonic plague (primary pneumonic plague). If treatment is not initiated within the first 24 h after symptoms appear, it is likely to be fatal within 48 h (18, 49). Septicemic plague can occur if Y. pestis gains direct access to the bloodstream via open wounds or fleabites (primary septicemic plague) (64) or as a result of spread from the lymphatic system to the bloodstream during advanced stages of bubonic plague (54). Y. pestis can also spread to the bloodstream and blood-filtering organs during late stages of pneumonic plague (39).In order for Y. pestis to cause disease, it must harbor the 70-kb pCD1 virulence plasmid, which encodes the Ysc type III secretion system (T3SS) and the Yop effector proteins (13, 69, 70). Yops inhibit phagocytosis by disrupting the actin cytoskeleton, diminish proinflammatory cytokine responses, and induce apoptosis of macrophages (13, 30, 50, 51, 54, 60, 62). In order for Yops to be delivered efficiently to host cells, adhesins must provide a docking function to facilitate T3S (8, 22, 59).Two adhesins shown to be important for Yop delivery in the related Yersinia species Y. enterocolitica and Y. pseudotuberculosis are YadA and invasin (Inv) (8, 47, 59). Y. pestis lacks both of these adhesins due to inactivation of inv by an IS1541 element and of yadA by a frameshift mutation (17, 52, 61, 65). Thus, we focused our studies on the Y. pestis adhesins described below.We recently identified Ail, an adhesin of Y. pestis that binds host cell fibronectin (73) and plays an important role in delivery of Yops to both phagocytic and nonphagocytic cells in vitro (22). In addition to having a defect in Yop delivery in vitro, a Y. pestis KIM5 Δail mutant has a >3,000-fold increased 50% lethal dose (LD₅₀) for mice in a septicemic plague model (22). However, the virulence defect of a KIM5 Δail mutant is not as severe as that of a KIM5 strain lacking the virulence plasmid (>10⁶-fold increase in LD₅₀) (22, 69). Thus, we set out to determine if there were other Y. pestis adhesins capable of facilitating Yop delivery.Four other adhesins of Y. pestis have been described. Plasminogen activator (Pla) is an adhesin and a protease. It can mediate binding to extracellular matrix proteins (32, 37, 43) and direct invasion of tissue culture cells (14, 36). Pla may also interact with the host cell receptor DEC-205 on macrophages and dendritic cells (80). Pla is known to be required for dissemination of a bubonic plague infection from the site of inoculation, presumably due to cleavage of fibrin clots by plasmin after Pla-mediated activation of plasminogen (27, 66). Its protease activity is also required for the development of fulminant pneumonic plague (39).pH 6 antigen (Psa) is a multifunctional surface structure that is induced at 37°C under low-pH conditions (4, 57, 75) and is controlled by the regulatory protein RovA (11). Psa can bind to β1-linked galactosyl residues in glycosphingolipids (53) and to phosphatidylcholine (26) on the surfaces of host cells. Due to its ability to bind surface receptors, Psa functions as an efficient adhesin (5, 24, 26, 41, 76). Psa-mediated adhesion also prevents phagocytosis (29). Finally, Psa can bind the Fc portion of human IgG (79) and interact with plasma low-density lipoproteins (LDLs) (44).The two remaining Y. pestis adhesins are the putative autotransporter YapC (23) and the pilus associated with the chaperone usher locus y0561 to y0563 (24). Neither locus is well expressed under laboratory conditions (23, 24; S. Felek and E. S. Krukonis, unpublished data), but when expressed in Escherichia coli, they can confer an increase in E. coli adhesion to several cultured cell lines (23, 24).Here we demonstrate that Ail, Pla, and Psa can mediate Yop delivery in vitro when expressed in a KIM5 mutant lacking all five adhesins (KIM5 Δ5), whereas overexpression of YapC or y0561 to y0563 does not allow for Yop delivery from the KIM5 Δ5 strain. Furthermore, we observed cumulative defects in virulence when each of three genes, ail, pla, and psaA, was deleted and tested in a mouse model. Thus, we have shown that all three adhesins can contribute to Yop delivery in vitro and to virulence in vivo.